Regulation of glutamine during mammalian or insect cell culture cultivation is of vital importance for optimization of cell growth and its productivity. Cell cultivation under depleted glutamine causes severe growth limitation, whereas increasing glutamine beyond a certain limit produces ammonia at toxic levels. Therefore it is critical to regulate glutamine during the course of cultivation processes. The determination of glutamine is also of importance in a clinical laboratory. Cerebrospinal glutamine levels are used with blood ammonia determinations in diagnosis of hepatic encephalopathy. Elevated glutamine levels are reported in parenteral nutrition, meningitis and in cerebral haemorrhage.
HPLC technique, commonly used for determination of glutamine, is time-consuming, expensive and requires skilled personnel.
U.S. Pat. No. 4,790,191 issued Oct. 25, 1988 to Romette et al. proposes an apparatus for measuring L-glutamine in a liquid sample. The apparatus includes a membrane on which are immobilized the enzymes glutaminase and glutamate oxidase. Glutamine in the sample is acted upon by the enzymes to form an enzymatic reaction product. The membrane is associated with a sensor, e.g. an oxygen electrode, which is capable of sensing either the product or a compound or element consumed or liberated in the process.
While the biosensor of the U.S. patent is useful, it has a drawback in that endogenous glutamic acid (also referred to in the literature as glutamate) present in the sample, i.e. cell culture medium, will interfere with the glutamine signal as the reaction of glutamine with glutaminase also yields glutamic acid. The sensor will therefore detect both glutamine and glutamic acid. In order to overcome this problem, a second measurement ("reference test") can be employed using immobilized glutamate oxidase alone as a reference analysis. This approach is both cumbersome and time-consuming since the membrane containing both immobilized glutamate oxidase and glutaminase (for determination of both glutamine and glutamic acid) and the membrane containing only immobilized glutamate oxidase (for determination of glutamate only) have to be interchanged during the course of measurement. In addition, this approach is only applicable for measurements in which the level of glutamine is significantly higher than that of glutamate (at least by the factor of ten). Further, oxygen based biosensors exhibit poor sensitivity due to their high current background (see Amperometric Biosensors, S. P. Hendry et al., Journal of Biotechnology, 15 (1990) 229-238). In this regard, hydrogen peroxide electrodes have been found superior to oxygen electrodes (Cattaneo et al., Monitoring Glutamine in Mammalian Cell Cultures Using an Amperometric Biosensor, Biosensors and Bioelectronics, 7 (1992) 329-334). However, endogenous glutamate interferes with the glutamine signal and a second measurement for the determination of glutamate is required.
It should be noted that a major disadvantage related to the use of hydrogen peroxide electrodes is the magnitude of the potential applied necessary for hydrogen peroxide measurement (+0.5 to +0.8 V, platinum vs. silver/silver chloride). Electroactive substances such as uric acid, ascorbic acid, acetaminophen etc. are known as potent interferents at this level. Such a drawback thus limits the widespread application of hydrogen-peroxide based biosensors for physiological samples or foodstuffs.
The determination of glucose levels in biological samples is an indispensable test for the diagnosis and therapy of certain illnesses, e.g. diabetes mellitus. The normal blood glucose level is about 90 mg/dL (5 mM) whereas the pathological value may increase up to 900 mg/dL (50 mM). Among several analytical procedures for the determination of glucose, electrochemical detection of enzymatically generated hydrogen peroxide is probably the most developed type of glucose biosensor. Amperometric glucose biosensors using immobilized glucose oxidase together with a sensitive hydrogen peroxide electrode have been used for in vitro and in vivo monitoring because of the high specificity of this enzyme for .beta.-D-glucose (Keilin et al., Biochem. J. 50, 1952, 331). In such a biosensor, the enzyme glucose oxidase catalyzes the oxidation of glucose to D-glucono-.delta.-lactone and hydrogen peroxide. The latter then contacts with a platinum anode vs silver/silver chloride cathode poised at +0.7 V where electrochemical oxidation takes place, and the current generated is directly proportional to the glucose concentration in the measured sample. Unfortunately, hydrogen peroxide amperometric detection is also sensitive to several naturally occurring electron donors, such as ascorbate, urate, acetaminophen, and so forth. Blood and urine contain significant concentrations of urate and ascorbate.
Among several methods proposed to improve the selectivity of the glucose biosensor against such electrochemically interfering substances, one solution is to form a differential system i.e to compensate the response by the addition of a second electrode not associated with glucose oxidase, see Clark, L. C., Biosensors: Fundamentals and Applications, Turner, Karube and Wilson, eds, Oxford Science Publications, 1987, Oxford, pp. 1-12. Another approach is described in U.S. Pat. No. 3,539,455 to Clark. It uses a permselective membrane (e.g. cellulose acetate) to cover the platinum anode. This type of membrane only allows the diffusion of small molecules such as oxygen or hydrogen peroxide, but excludes ascorbate and other large-particle potential interfering substances. The main disadvantage of this approach is that it creates an additional diffusion layer that adversely affects the sensitivity and the response of the enzyme electrode.